Chun Te Chen

p53 regulates epithelial–mesenchymal transition and stem cell properties through modulating miRNAs

The epithelial–mesenchymal transition (EMT) has recently been linked to stem cell phenotype. However, the molecular mechanism underlying EMT and regulation of stemness remains elusive. Here, using genomic approaches, we show that tumour suppressor p53 has a role in regulating both EMT and EMT-associated stem cell properties through transcriptional activation of the microRNA miR-200c. p53 transactivates miR-200c through direct binding to the miR-200c promoter. Loss of p53 in mammary epithelial cells leads to decreased expression of miR-200c and activates the EMT programme, accompanied by an increased mammary stem cell population. Re-expressing miR-200c suppresses genes that mediate EMT and stemness properties and thereby reverts the mesenchymal and stem-cell-like phenotype caused by loss of p53 to a differentiated epithelial cell phenotype. Furthermore, loss of p53 correlates with a decrease in the level of miR-200c, but an increase in the expression of EMT and stemness markers, and development of a high tumour grade in a cohort of breast tumours. This study elucidates a role for p53 in regulating EMT–MET (mesenchymal–epithelial transition) and stemness or differentiation plasticity, and reveals a potential therapeutic implication to suppress EMT-associated cancer stem cells through activation of the p53–miR-200c pathway.

IKKβ Suppression of TSC1 Links Inflammation and Tumor Angiogenesis via the mTOR Pathway

TNFalpha has recently emerged as a regulator linking inflammation to cancer pathogenesis, but the detailed cellular and molecular mechanisms underlying this link remain to be elucidated. The tuberous sclerosis 1 (TSC1)/TSC2 tumor suppressor complex serves as a repressor of the mTOR pathway, and disruption of TSC1/TSC2 complex function may contribute to tumorigenesis. Here we show that IKKbeta, a major downstream kinase in the TNFalpha signaling pathway, physically interacts with and phosphorylates TSC1 at Ser487 and Ser511, resulting in suppression of TSC1. The IKKbeta-mediated TSC1 suppression activates the mTOR pathway, enhances angiogenesis, and results in tumor development. We further find that expression of activated IKKbeta is associated with TSC1 Ser511 phosphorylation and VEGF production in multiple tumor types and correlates with poor clinical outcome of breast cancer patients. Our findings identify a pathway that is critical for inflammation-mediated tumor angiogenesis and may provide a target for clinical intervention in human cancer.

Degradation of Mcl-1 by β-TrCP Mediates Glycogen Synthase Kinase 3-Induced Tumor Suppression and Chemosensitization

ABSTRACT Apoptosis is critical for embryonic development, tissue homeostasis, and tumorigenesis and is determined largely by the Bcl-2 family of antiapoptotic and prosurvival regulators. Here, we report that glycogen synthase kinase 3 (GSK-3) was required for Mcl-1 degradation, and we identified a novel mechanism for proteasome-mediated Mcl-1 turnover in which GSK-3β associates with and phosphorylates Mcl-1 at one consensus motif (155STDG159SLPS163T; phosphorylation sites are in italics), which will lead to the association of Mcl-1 with the E3 ligase β-TrCP, and β-TrCP then facilitates the ubiquitination and degradation of phosphorylated Mcl-1. A variant of Mcl-1 (Mcl-1-3A), which abolishes the phosphorylations by GSK-3β and then cannot be ubiquitinated by β-TrCP, is much more stable than wild-type Mcl-1 and able to block the proapoptotic function of GSK-3β and enhance chemoresistance. Our results indicate that the turnover of Mcl-1 by β-TrCP is an essential mechanism for GSK-3β-induced apoptosis and contributes to GSK-3β-mediated tumor suppression and chemosensitization.

Tyrosine phosphorylation controls PCNA function through protein stability.

The proliferating cell nuclear antigen (PCNA) is an essential protein for DNA replication and damage repair. How its function is controlled remains an important question. Here, we show that the chromatin-bound PCNA protein is phosphorylated on Tyr 211, which is required for maintaining its function on chromatin and is dependent on the tyrosine kinase activity of EGF receptor (EGFR) in the nucleus. Phosphorylation on Tyr 211 by EGFR stabilizes chromatin-bound PCNA protein and associated functions. Consistently, increased PCNA Tyr 211 phosphorylation coincides with pronounced cell proliferation, and is better correlated with poor survival of breast cancer patients, as well as nuclear EGFR in tumours, than is the total PCNA level. These results identify a novel nuclear mechanism linking tyrosine kinase receptor function with the regulation of the PCNA sliding clamp.

EZH2 Promotes Expansion of Breast Tumor Initiating Cells through Activation of RAF1-β-Catenin Signaling

It has been proposed that an aggressive secondary cancer stem cell population arises from a primary cancer stem cell population through acquisition of additional genetic mutations and drives cancer progression. Overexpression of Polycomb protein EZH2, essential in stem cell self-renewal, has been linked to breast cancer progression. However, critical mechanism linking increased EZH2 expression to BTIC (breast tumor initiating cell) regulation and cancer progression remains unclear. Here, we identify a mechanism in which EZH2 expression-mediated downregulation of DNA damage repair leads to accumulation of recurrent RAF1 gene amplification in BTICs, which activates p-ERK-β-catenin signaling to promote BTIC expansion. We further reveal that AZD6244, a clinical trial drug that inhibits RAF1-ERK signaling, could prevent breast cancer progression by eliminating BTICs.

KEAP1 E3 Ligase-Mediated Downregulation of NF-κB Signaling by Targeting IKKβ

IkappaB kinase beta (IKKbeta) is involved in tumor development and progression through activation of the nuclear factor (NF)-kappaB pathway. However, the molecular mechanism that regulates IKKbeta degradation remains largely unknown. Here, we show that a Cullin 3 (CUL3)-based ubiquitin ligase, Kelch-like ECH-associated protein 1 (KEAP1), is responsible for IKKbeta ubiquitination. Depletion of KEAP1 led to the accumulation and stabilization of IKKbeta and to upregulation of NF-kappaB-derived tumor angiogenic factors. A systematic analysis of the CUL3, KEAP1, and RBX1 genomic loci revealed a high percentage of genome loss and missense mutations in human cancers that failed to facilitate IKKbeta degradation. Our results suggest that the dysregulation of KEAP1-mediated IKKbeta ubiquitination may contribute to tumorigenesis.

Myeloid cell leukemia-1 inversely correlates with glycogen synthase kinase-3β activity and associates with poor prognosis in human breast cancer

Myeloid cell leukemia-1 (Mcl-1), an antiapoptotic Bcl-2 family member, is overexpressed in many types of human cancer and associates with cell immortalization, malignant transformation, and chemoresistance. Glycogen synthase kinase-3beta (GSK-3beta), a key component of the Wnt signaling pathway, is involved in multiple physiologic processes such as protein synthesis, tumorigenesis, and apoptosis. Here, we report that expression of Mcl-1 was correlated with phosphorylated GSK-3beta (p-GSK-3beta) at Ser(9) (an inactivated form of GSK-3beta) in multiple cancer cell lines and primary human cancer samples. In addition, Mcl-1 was strikingly linked with poor prognosis of human breast cancer, in which the high level of Mcl-1 was related to high tumor grade and poor survival of breast cancer patients. Furthermore, we found that activation of GSK-3beta could down-regulate Mcl-1 and was required for proteasome-mediated Mcl-1 degradation. Under some physiologic conditions, such as UV irradiation, anticancer drug treatment, and inhibition of growth factor pathways, Mcl-1 was down-regulated through activation of GSK-3beta. Our results indicate that Mcl-1 stabilization by GSK-3beta inactivation could be involved in tumorigenesis and serve as a useful prognostic marker for human breast cancer.

Emodin Down-Regulates Androgen Receptor and Inhibits Prostate Cancer Cell Growth

Hormone-refractory relapse is an inevitable and lethal event for advanced prostate cancer patients after hormone deprivation. A growing body of evidence indicates that hormone deprivation may promote this aggressive prostate cancer phenotype. Notably, androgen receptor (AR) not only mediates the effect of androgen on the tumor initiation but also plays the major role in the relapse transition. This provides a strong rationale for searching new effective agents targeting the down-regulation of AR to treat or prevent advanced prostate cancer progression. Here, we show that emodin, a natural compound, can directly target AR to suppress prostate cancer cell growth in vitro and prolong the survival of C3(1)/SV40 transgenic mice in vivo. Emodin treatment resulted in repressing androgen-dependent transactivation of AR by inhibiting AR nuclear translocation. Emodin decreased the association of AR and heat shock protein 90 and increased the association of AR and MDM2, which in turn induces AR degradation through proteasome-mediated pathway in a ligand-independent manner. Our work indicates a new mechanism for the emodin-mediated anticancer effect and justifies further investigation of emodin as a therapeutic and preventive agent for prostate cancer.

ARD1 Stabilization of TSC2 Suppresses Tumorigenesis Through the mTOR Signaling Pathway

Acetylation by ARD1 of an inhibitor of mammalian target of rapamycin may inhibit cancer growth. Inhibiting mTOR and Tumor Growth The acetylase arrest-defective protein 1 (ARD1) has been implicated in mammalian cell proliferation and apoptosis, and the dysfunction of these processes contributes to cancer development. Kuo et al. searched an online database for a possible relationship between ARD1 and cancer and found that increased ARD1 mRNA abundance was associated with various measures of improved clinical outcome in patients with breast cancer, suggesting that ARD1 might act as a tumor suppressor. They uncovered ARD1 loss of heterozygosity (LOH) in primary breast cancer and several different cancer cell lines. Further analysis indicated that ARD1 acetylated—and thereby stabilized—tuberous sclerosis 2 (TSC2), part of a complex that inhibits signaling through the mammalian target of rapamycin (mTOR) pathway, a master regulator of cellular metabolism. ARD1-dependent inhibition of mTOR signaling decreased cell proliferation and increased autophagy, thereby suppressing tumor growth in nude mice. Moreover, ARD1 abundance correlated with that of TSC2 in various types of cancer. The authors thus propose that ARD1 suppressed development of multiple types of cancer through its stabilization of TSC2 and the consequent inhibition of mTOR signaling. Mammalian target of rapamycin (mTOR) regulates various cellular functions, including tumorigenesis, and is inhibited by the tuberous sclerosis 1 (TSC1)–TSC2 complex. Here, we demonstrate that arrest-defective protein 1 (ARD1) physically interacts with, acetylates, and stabilizes TSC2, thereby repressing mTOR activity. The inhibition of mTOR by ARD1 inhibits cell proliferation and increases autophagy, thereby inhibiting tumorigenicity. Correlation between ARD1 and TSC2 abundance was apparent in multiple tumor types. Moreover, evaluation of loss of heterozygosity at Xq28 revealed allelic loss in 31% of tested breast cancer cell lines and tumor samples. Together, our findings suggest that ARD1 functions as an inhibitor of the mTOR pathway and that dysregulation of the ARD1-TSC2-mTOR axis may contribute to cancer development.

IKKα Activation of NOTCH Links Tumorigenesis via FOXA2 Suppression

Proinflammatory cytokine TNFα plays critical roles in promoting malignant cell proliferation, angiogenesis, and tumor metastasis in many cancers. However, the mechanism of TNFα-mediated tumor development remains unclear. Here, we show that IKKα, an important downstream kinase of TNFα, interacts with and phosphorylates FOXA2 at S107/S111, thereby suppressing FOXA2 transactivation activity and leading to decreased NUMB expression, and further activates the downstream NOTCH pathway and promotes cell proliferation and tumorigenesis. Moreover, we found that levels of IKKα, pFOXA2 (S107/111), and activated NOTCH1 were significantly higher in hepatocellular carcinoma tumors than in normal liver tissues and that pFOXA2 (S107/111) expression was positively correlated with IKKα and activated NOTCH1 expression in tumor tissues. Therefore, dysregulation of NUMB-mediated suppression of NOTCH1 by TNFα/IKKα-associated FOXA2 inhibition likely contributes to inflammation-mediated cancer pathogenesis. Here, we report a TNFα/IKKα/FOXA2/NUMB/NOTCH1 pathway that is critical for inflammation-mediated tumorigenesis and may provide a target for clinical intervention in human cancer.